The present invention relates to a method for recovery of bioactive colostrum components. More precisely the present invention is directed to a method for treating colostrum to reduce the bioburden while retaining a high active protein content. The invention is also directed to the colostrum treated by said method and to the use thereof.
Milk produced just after parturition is called colostrum. This particular milk contains about 20 times more protein than milk produced later. Colostrum is, therefore, an excellent source of many valuable proteins, such as biologically active proteins like growth factors and especially immunoglobulins. The colostrum can, therefore, be used as a source of said valuable proteins e.g. in food or clinical preparations. However, colostrum is often contaminated with a high amount of bacteria and other cell material, which is not allowed in a product qualified as a food or clinical product.
The conventional way of reducing the bioburden of milk is pasteurization and ultra-heat treatment i.e. the milk is exposed to heat for a short period of time. However, the heat treatment does not only destroy the microorganisms present in the milk, but also denatures the valuable biologically active proteins. Colostrum is especially unsuitable for heat treatment, as the high protein content makes it coagulate at elevated temperatures. A method of reducing the bioburden of colostrum by centrifugation has been described in W097 /16977. However, an effective reduction of bacteria requires such a high force of gravitation that proteins might precipitate together with other particles present in a protein rich solution.
Other methods of reducing microbial contaminants in milk are gamma radiation (U.S. Pat. No. 4,784,850) and treatment with xcex2-propiolactone (U.S. Pat. No. 3,911,108). Also, these methods tend to denature proteins to some extent. Sterile filtration is still another method of removing microbes from milk (U.S. Pat. No. 5,256,437; U.S. Pat. No. 5,683,733; Pedersen P. J., (1991) IDF special issue no 9201. Microfiltration for the Reduction of Bacteria in Milk and Brine, In New Applications of Membrane Processes, 33-50; Osterland N., New Developments in Membrane Processing, IDF 25th International Dairy Congress Sep. 21-24, 1998 xc3x85rhus, Denmark; and Rosenberg M. (1995), Trends in Food Science and Technology, 6:12-19). Filtration has also been used for separating different components in milk such as skim milk and cream-enriched fractions (U.S. Pat. No. 4,140,806), and dissolved and undissolved components in milk (U.S. Pat. No. 5,028,436). Filtration does not usually substantially affect the proteins, but the filters rapidly foul. This is especially a problem with protein rich colostrum, where the casein easily clogs the filters.
The problem with clogged filters has previously been solved by partially or completely removing casein from the colostrum, and/or by diluting the colostrum before filtration. Casein can be removed by either acid or enzymatic precipitation and centrifugation to obtain whey (U.S. Pat. No. 4,644,056 and GB 1,573,995). U.S. Pat. No. 5,670,196 discloses a method of microfiltrering colostrum, whereby defatted colostrum is first acidified to precipitate casein, which is removed by centrifugation, and then the whey is filtered through a charged depth filter to reduce the microorganism content. U.S. Pat. No. 5,707,678 is directed to a similar method, where casein is removed, after which the acidified whey is first ultrafiltered and then microfiltered. The main drawback of these methods is that large amounts of valuable antibodies and other proteins tend to precipitate together with the casein. In addition the removal of casein is a laborious, time consuming and expensive process.
U.S. 5,147,548 discloses a method of sterile-filtering colostrum without previously removing the casein. The optionally defatted colostrum is acidified to a pH of less than 3.5. The casein precipitates at a pH of 5 to 4, but it returns into solution as the pH continues to drop. The acidic solution was found to differ so extensively from the original colostrum that it could be sterile filtered either as such or after neutralizing it back to its original pH. The filter used is a depth filter or a membrane filter. In a preferred embodiment the colostrum is diluted into a sodium chloride solution prior to acidifying. However, also this method has drawbacks. The immunoglobulins are easily inactivated at low pH. Further, the casein precipitation is not fully reversible resulting also in protein loss, and the dilution of the colostrum increases process time and expenses.
The object of the present invention is to provide a simple, effective and economic method of reducing the bioburden of colostrum without substantially affecting the proteins contained therein. The method provides the elimination of microbial contaminants without substantial loss of the high and versatile biologically active protein content and/or activity. The method thus enables effective reduction of the bioburden while retaining the maximum immunoglobulin activity, especially IgG. No previous precipitation of casein nor any dilution, or addition of salts/acids/bases or other chemicals are needed, and there is no temperature denaturation of the antibodies present.
Another object of the invention is to provide a colostrum preparation of high hygienic standard, which qualifies as a food or clinical article. The colostrum preparation can be used in the form of a beverage or food or in dry form for promoting health or for treating or preventing disorders, which can be cured by immunoglobulins or additional colostrum proteins.
It has surprisingly been found, that a simple filtering system allows the reduction of the bioburden of colostrum without multistep pretreatment and loss of protein activity. The objects of the present invention can thus be achieved by a method for treating colostrum, which method is characterized by
(a) collecting colostrum
(b) defattening said colostrum
(c) carrying out cross flow microfiltration (CFMF) of the defatted colostrum using a tangential flow filter (TFF) device with open channels, and a filter having a pore size of 0.1-0.5 xcexcm, and
(d) recovering the filtrate.
The colostrum of the present invention is characterized by being treated by the method of the invention.
The invention is further directed to the use of the treated colostrum for the manufacture of clinical nutritive preparations, functional foods, or food supplements comprising said colostrum.
According to the method of the present invention colostrum is collected from a mammal, which can be any mammal e.g. goat or sheep, but preferably it is a cow. Preferably the mammal has previously been immunized or hyperimmunized against a pathogen, whereby colostrum useful for treating or preventing the disease caused by the pathogen can be obtained. The colostrum is collected soon after parturition, when the IgG content is at its maximum, usually within three days and preferably within 48 hours from parturition. The colostrum is normally, but not necessarily frozen and then cautiously thawed before processing, whereby high temperatures should be avoided. The fat is separated from the colostrum in any conventional way, usually by centrifugation. Preferably the obtained skim milk is then clarified for example by prefiltration through a depth filter or a membrane filter to remove possible clumps prior to the cross flow microfiltration step. Suitable filter media are e.g. polypropylene, regenerated cellulose or polyethersulfone having a pore size of 0,1-150 xcexcm, normally about 0,5-50 xcexcm.
Microfiltration (MF) is a pressure-driven separation process that uses membranes of a given pore size to separate components in a solution or suspension on the basis of their size difference. Although larger particles can be removed by use of non-membrane or depth filters, only a membrane filter having a precisely defined pore size can ensure quantitative retention. The conventional MF is a dead-end process, where the solution is vertically passed through the membrane. The particles which are too big to pass the pores are retained on the membrane surface, whereby the filter is quickly clogged. A development of MF is cross-flow microfiltration (CFMF), where the retained solution circulates tangentially across the membrane surface. Cross-flow is the rate at which the material flows across the membrane surface and is important as it generates a number of forces which tend to remove the deposited layers from the membrane surface thus helping to keep the membrane clean.
In cross flow microfiltration permeate or filtrate is the solution, which has passed through the membrane, retentate is the solution or suspension retained by the membrane and flux is the filtrate flow through the membrane.
The microfiltration of the defatted colostrum according to the present invention is carried out by cross flow microfiltration (CFMF) using a tangential flow filter (TFF) device. TFF membrane devices may be linear or turbulence promoted depending on the tangential flow channel design for material flow. So called open channel devices have straight, open feed flow channels, which allow a laminar flow in the channels, whereas the turbulence promoted so called thin channel devices have feed flow channels containing e.g. a screen, which promotes turbulence. The open channel devices should be used in the method of the present invention. TFF open channel devices may be obtained e.g. from Millipore Corp., Bedford, Mass., USA (Prostak(trademark)).
The membrane to be used in the open channel device is a flat sheet membrane, which has a pore size preventing bacteria and other microorganisms from passing to the filtrate, but allowing the desired proteins e.g. IgG to penetrate. A suitable pore size for this purpose is normally between 0.1-0.5 xcexcm, and preferably 0.2-0.45 xcexcm, especially about 0.2 xcexcm. The filters can be e.g. polysulfone, cellulose, or especially fluorocarbon polymer based membranes. Polyvinylidene fluoride (PVDF) membranes are especially suitable for the CFMF of colostrum, and most preferred are PVDF membranes, which have been hydrophilized.
In order to increase the filter area and speed up the filtrate flux several filter device modules can be combined. Normally 100-200 liter colostrum is filtered with a capacity of 10-50 l/m2h, preferably 20-40 l/m2h and especially about 25 l/m2h at a pressure of about 0.5-3 bar, preferably 0.8-2 bar and especially 1 bar. The filtrate containing the active proteins, but substantially free of any bacterial or other microbial contaminants, is recovered. Optionally the cross-flow microfiltered colostrum is finally sterile filtered by conventional microfiltration through a 0.2-0.45 xcexcm membrane to ensure a sterile end product.
Often it is desirable to concentrate the filtered colostrum to enrich the proteins in question and optionally to remove salts. This can be done in a way known per se e.g. by ultrafiltration or reverse osmosis depending on the nature of the protein. It is finally possible to dry the filtered colostrum for example by lyophilization. Alternatively, the colostrum may be spray dried under controlled temperatures to avoid protein denaturation. The dried colostrum can either be e.g. encapsulated and used as such or dissolved in an aqueous solution before use.
In order to keep the microbial count low, the process should be carried out at low temperature. For the fat separation a temperature of about 40xc2x0 C. is convenient, but the rest of the process is carried out at lower temperatures, preferably not exceeding 15xc2x0 C., and mainly at a temperature of 2-10xc2x0 C. Even at pumping during the CFMF, the temperature can be kept low and never allowed to exceed 40xc2x0 C.
The method of the present invention may be used for different purposes where the aim is to obtain microbial free colostrum without substantial loss of valuable protein activities. Colostrum contains a lot of biologically active proteins such as hormones, growth factors, lactoferrin, bactericidal proteins and especially antibodies i.e. immunoglobulins of the classes IgG, IgA and IgM.
Colostrum of the present invention can be used in dry form or in the form of a beverage or food. It is especially suitable for the manufacture of clinical nutritive preparations, functional foods and food supplements comprising the treated colostrum. A clinical nutritive preparation is an article that is suitable for a person having special medical needs for the active components therein. Functional foods are foods having a health promoting effect, and food supplement is a food additive, which is added to give the food desirable properties. These products can conveniently be given orally to subjects in need thereof. IgG for example is useful in protecting mucous membranes against pathogen colonization and penetration, and it is especially suitable for treating or preventing enteropathogenic infections. The IgG rich, microbe free colostrum of the present invention may e.g. be given to immunosuppressed patients.